Eukaryotic cell cycle progression is mediated by the precise oscillations of cyclin-dependent kinase (CDK) activity, which controls the periodicity of DNA replication and mitosis. CDKs are activated by cyclins and inactivated by CDK inhibitors (CKIs). Altered expression of cyclins or CKIs may lead to increased CDK activity and thus uncontrolled cell proliferation and ultimately cancer. Cyclin and CKI levels can be regulated post-translationally by ubiquitin-proteasome dependent proteolysis. In fact, ubiquitin-proteasome dependent degradation of cyclins and CKIs has recently been shown to regulate two cell cycle transitions: G1-S and mitosis. Ubiquitin-mediated proteolysis involves assembly of a ubiquitin chain on a substrate followed by degradation of the substrate by the 26S proteasome. Substrate specificity for ubiquitination is mediated by ubiquitin ligases or E3s. The E3 ubiquitin ligase for the G1-S transition is the Skp1p-cullin-F-box complex. Cyclins and CKIs involved in the G1-S transition that are known to undergo ubiquitin-proteasome dependent degradation include cyclin E and p21Cip1. However, the E3 ubiquitin ligases that target these cell cycle regulators for degradation are not known. Recent work here in the lab of Dr. Roberts has demonstrated that the human cullin CUL-3 binds to cyclin E and promotes the formation of ubiquitin chains on cyclin E. Similarly, CUL-3 binds to p21Cip1 and promotes the ubiquitination of p21Cip1. The primary objectives of this proposal are 1) to determine the proteins that interact with CUL-3 to create an E3 ubiquitin ligase and 2) to understand the mechanism by which CUL-3 complexes may regulate cyclin E and p21Cip1. Three approaches that will be used to identify CUL-3 interacting proteins include 1) mass spectrometric analysis of CUL-3 complexes, 2) a two-hybrid screen using CUL-3 as the bait, and 3) binding studies of CUL-3 to known ubiquitin conjugating enzymes (E2s). The functional characterization of CUL-3 and its interacting proteins will be facilitated by reconstituting CUL-3 dependent ubiquitination of cyclin E and p21Cip1 both in vivo and in vitro.